There is an increasing interest in so-called “lab-on-a-chip” technologies for cell-based, scientific, and/or clinical applications. Such techniques can integrate various procedures on small devices, including, e.g., primary sample collection, sample processing, and/or data analysis. Lab-on-a-chip devices often use microfluidic structures to process small samples and can facilitate more rapid processing of samples. Such devices and methods can often be used without a need for extensive lab support and thus may be well suited for field and point-of-care applications.
Migration of biological species in response to certain environmental factors can be very important in understanding various mechanisms that occur in the body and in developing effective treatments for various conditions. Neutrophil directional migration in response to soluble chemoattractant gradients is a critical process during the response of the innate immune against bacterial infection. Also known as chemotaxis, it plays a significant role in several other physiological processes such as inflammation, wound healing, metastasis, atherosclerosis, arthritis etc. With recent developments in microfluidic technologies, cell migration research has gained significant attention in recent years and a variety of in vitro migration assays have been developed. One major challenge for these assays is the isolation of neutrophils from whole blood without activating them or altering their phenotype. The most commonly used isolation procedures involve Ficoll-Hypaque centrifugation, erythrocyte lysis or the combination of these two techniques. Alternatively, neutrophils can be separated from whole blood by negative immunomagnetic separation techniques which deplete all unwanted cells by selective capture on functionalized magnetic beads. These techniques require large volumes of whole blood (of the order of milliliters) and are prone to mechanical, osmotic or thermal shocks to which neutrophils are highly sensitive. While recent studies involving high speed microfluidic lysis process can yield 98-100% leukocytes with minimal activation, the method still requires a centrifugation step for debris removal, which is difficult to implement on the chip. The effects of cell lysate contaminat on neutrophils' responsiveness to subsequent chemotactic gradients are also unknown.
A second critical challenge in studying neutrophil chemotaxis is establishing more complex gradient generation schemes to study sequential effects of different chemokines or activity of anti-inflammatory agents. Since the development of the Boyden chamber in 1962, several other techniques including micropipette generated gradients, hydrogel based assay, Zigmond chamber and Dunn chamber have been proposed for studying chemotactic responsiveness of many cells, including neutrophils, to artificial chemical gradients. However, many of these assays pose limitations of gradient nonlinearity and spatial/temporal instability making repeatability of the experimental conditions difficult to control. While some of these methods are unsuitable for observing single cell responses (e.g., Boyden chamber), others lack the capability to support multifactor combinatorial gradients or only offer short lived gradients (e.g., Dunn chamber). More recently, microfluidic gradient generators have greatly evolved and a number of models categorized as parallel-flow or flow-resistive gradient generators have been proposed to perform in vitro chemotaxis. Although many of the microfluidic techniques succeed in eliminating the issues related to quantification as well as spatial and temporal stability of gradients, they fail either to provide dynamic control over the established gradients or to independently generate combinatorial gradients of multiple chemokines. Moreover, none of these techniques were able to provide a complete assay for combined capture and migration analysis without undergoing additional isolation steps.
The directed migration of neutrophils in vivo involves sequential signaling events to guide them from blood stream into the tissue near the site of injury. The endothelium, when exposed to inflammation signals, may express cell adhesion molecules, such as P-selectin and E-selectin, and can present chemoattractants, such as IL-8, for which complementary receptors exist on leukocyte surface. This mechanism can lead to localization of circulating neutrophils towards the inner wall of blood vessels where hemodynamic shear stresses may be smaller, and the cells can begin to roll and migrate based on, e.g., weak transient binding of CAMs with cell receptors. The presence of inflammation signals on the endothelial cells may later induce the expression of integrin receptors, which can lead to cellular arrest followed by transmigration of neutrophils into the tissue.
The commonly used neutrophils isolation methods such as Ficoll-Hypaque isolation and immunomagnetic separation work reliably, however, they require over an hour to isolate neutrophils and demand several milliliters of whole blood. These methods are not suited for studies where rat or mice models are used due to the lower volumes of blood samples available in these smaller animals (only a few milliliters). Such experiments often mandate sacrificing the animal in order to extract enough blood, and make prolonged monitoring of the animals very difficult to accomplish. Recently, microfluidic devices coated with antibodies have been demonstrated for the isolation of leukocytes from whole blood. These devices require only small volumes of blood and accomplish the isolation of targeted leukocyte subpopulation in less than 10 minutes. One limitation of these devices, in the context of the chemotaxis assays, is that the binding of the cells to the antibody coated surface is strong and practically irreversible, prohibiting their use for chemotaxis assays.
The ability to visualize moving cells in a chemotaxis assay can be important for differentiating between chemotaxis and chemokinesis (e.g., directional vs. random motility). Further, visualization of cell morphology changes upon exposure of cells to chemokines and lipid mediators may also be important in understanding the mechanisms by which such substances can affect cell migration. Conventional clinical chemotaxis assays (e.g., Boyden chambers) may not provide direct visualization of cells.
Microfluidic gradient generators have been developed or proposed using gradient techniques such as, e.g., parallel-flow and flow-resistive gradient generators to perform in vitro chemotaxis. Such microfluidics devices and techniques are described, e.g., in T. M. Keenan and A. Folch, Lab on a Chip, 2008, 8, 34-57; D. Irimia et al., Lab on a Chip, 2006, 6, 191-198; T. M. Keenan et al., Applied Physics Letters, 2006, 89; and R. M. Kuntz and W. M. Saltzman, Biophysical Journal, 1997, 72, 1472-1480. Although some of these techniques may improve performance issues relating to spatial and temporal stabilities of gradients, they generally do not provide dynamic control over established gradients and/or allow independent, combinatorial gradients of multiple chemokines or other materials to be generated. Moreover, none of these techniques or devices provides a complete assay for migration analysis, optionally combined with capture techniques, without performing additional isolation steps.
Accordingly, there is a need for improved methods and devices for quantitative analysis of migration behavior in biological samples.